Method of preventing the development of melanoma

ABSTRACT

A method for treating melanoma or preventing the development of melanoma comprising administration of a composition comprising  64 Zn e (Asp) 2  in a therapeutically effective amount. Such administration may be via injection such as intratumoral and/or intravenous injection and may be once a day or more than once a day. A composition for the treatment of or prevention of melanoma comprising  64 Zn e (Asp) 2  in a therapeutically effective amount. The composition may be a liquid suitable for injection.

TECHNICAL FIELD

This disclosure relates to oncology, pharmacology and veterinarymedicine and relates specifically to treating or preventing malignantskin diseases such as melanoma.

BACKGROUND

Melanoma is a malignant tumor that develops from melanocytes(pigment-containing cells that produce melanin), which are predominantlylocated in the basal layer of the skin's epidermis and the middle layerof the eye (Hurst E A et al., Archives of Dermatology Research, 2003,139: 1067-1073). This type of pathology accounts for 10 percent of allmalignant skin lesions. Its annual incidence rate is 5%. Starting fromthe 1940s, the incidence of melanoma has doubled every year. Melanoma isthe sixth most common cancer among men and the seventh most commoncancer in women. The average incidence rates for skin melanoma vary from3-5 cases per 100,000 people per year in the Mediterranean countries to12-20 cases per 100 thousand people per year in the Nordic countries andcontinue to grow. The death rate is 2-3 cases per 100 thousand peopleevery year with slight changes depending on the geographical location,and has remained relatively stable over the past decade. An increasedexposure to ultraviolet radiation of a genetically predisposedpopulation, at least in part, results in a steady increase in melanomaincidence over the past decades (Oncology Clinical Practice Guidelinesof the European Society for Medical Oncology (ESMO), 2010, p. 294-300).Malignant melanoma is responsible for 60-80% of deaths from skin cancersand its five-year survival rate is 14%. In the United States, 2% of thepopulation was diagnosed with this type of skin cancer, which is a causeof approximately 10,000 deaths every year. At the same time melanoma isa tumor with an extremely high potential of systemic metastases.

Yet, possibilities for treating or preventing melanoma are limited.

SUMMARY

In one aspect, a method is provided of preventing the development ofmelanoma comprising intratumoral and/or intravenous administration to asubject of ⁶⁴Zn_(e)(Asp)₂, containing 2 molecules of aspartic acid foreach molecule of zinc, at a therapeutically effective dose. The non-⁶⁴Zn-enriched form is known as zinc di-aspartate and has a molecularformula Zn(C₄H₆NO₄)₂. In certain embodiments, the aspartate of⁶⁴Zn_(e)(Asp)₂ is enriched for the L-enantiomer. In further embodiments,it is at least 90% L-enantiomer, at least 95% L-enantiomer, or least 98%L-enantiomer.

In another aspect, a method is provided of preventing melanomametastasis comprising intratumoral and/or intravenous administration toa subject of ⁶⁴Zn_(e)(Asp)₂, containing 2 molecules of aspartic acid foreach molecule of zinc, at a therapeutically effective dose.

In yet another aspect, a composition is provided for use in preventingthe development of melanoma and/or in preventing melanoma metastasiscomprising ⁶⁴Zn_(e)(Asp)₂ in a therapeutically effective amount and atleast one carrier or excipient.

In each of the above aspects, in certain embodiments, the Zn-enrichedzinc of the ⁶⁴Zn_(e)(Asp)₂ is at least 80% ⁶⁴Zn, at least 85% ⁶⁴Zn, atleast 90% ⁶⁴Zn, at least 95% ⁶⁴Zn, or at least 99% ⁶⁴Zn. Examples ofsuitable levels of ⁶⁴Zn enrichment include any specific value within therecited ranges, such as 80%, 85%, 90%, 95%, 99%, and 99.8% ⁶⁴Zn. As usedherein, unless otherwise indicated, X % ⁶⁴Zn means that, out of 100 zincatoms, X is ⁶⁴Zn. For example, in zinc that is 95% ⁶⁴Zn, 95% of theatoms are ⁶⁴Zn. Unless otherwise indicated, the term “⁶⁴Zn_(e)” is usedherein as shorthand for “⁶⁴Zn-enriched zinc”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows survival data for experimental animals (% vs. control) thatwere given intratumoral (“i/t”) injections of ⁶⁴Zn_(e)(Asp)₂ comprising2 molecules of aspartic acid/atom of zinc (written in shorthand hereinas “⁶⁴Zn_(e)(Asp)₂”, unless otherwise indicated) at day 5 aftertransplantation of B16 melanoma cells into mice. Data on survival in thegroup treated with ⁶⁴Zn_(e)(Asp)₂ (intra-tumor administration of⁶⁴Zn_(e)(Asp)₂ at 5th day after tumor transplantation) are compared withthose in the control group.

FIG. 2 shows data on the antitumor activity of ⁶⁴Zn_(e)(Asp)₂ in mice(mean tumor volume in mm³) that were given intratumoral injections of⁶⁴Zn_(e)(Asp)₂ at day 5 after transplantation of B16 melanoma cellscompared to the control group.

FIG. 3 shows data on the kinetics of B16 melanoma growth in mice (meantumor volume in mm³) that were given intratumoral injections of⁶⁴Zn_(e)(Asp)₂ at day 5 after transplantation of B16 melanoma cellscompared to the control group.

FIGS. 4A-4C shows data on the inhibition of metastatic process in thelungs in C57Bl mice transplanted with B16 melanoma after intravenousadministration of ⁶⁴Zn_(e)(Asp)₂ 45 minutes and 24 hours aftertransplantation of the tumor cells. FIG. 4A—control, FIG. 4B—intravenousinjection of ⁶⁴Zn_(e)(Asp)₂ 24 hours after transplantation of tumorcells, FIG. 4C—intravenous administration of ⁶⁴Zn_(e)(Asp)₂ 45 minutesafter transplantation of tumor cells.

FIG. 5A-FIG. 5B show data on the quantitative evaluation of metastaticactivity of melanoma cells after intravenous administration of⁶⁴Zn_(e)(Asp)₂ 45 minutes and 24 hours after transplantation of tumorcells, represented as the mean number of metastases (FIG. 5A) and thepercentage of inhibitions of metastatic activity (FIG. 5B).

DETAILED DESCRIPTION

As used herein, the word “a” or “plurality” before a noun represents oneor more of the particular noun.

For the terms “for example” and “such as,” and grammatical equivalencesthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. As used herein, the term “about” ismeant to account for variations due to experimental error. Allmeasurements reported herein are understood to be modified by the term“about,” whether or not the term is explicitly used, unless explicitlystated otherwise. As used herein, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

The term “treating” as used herein with respect to a medical conditionsuch as melanoma means diminishing the severity and/or consequences ofthe condition, slowing the progression of the condition, preventing thespread of the condition in a patient who has the condition, preventingmetastasis of the condition, at least substantially, and/or curing thecondition.

The term “preventing” as used herein with respect to a medical conditionsuch as melanoma means preventing the development of the condition, atleast substantially, diminishing the severity and/or consequences of thecondition, slowing the progression of the condition, preventing thespread of the condition in a patient who has the condition, and/orpreventing metastasis of the condition, at least substantially.

“Effective amount,” “prophylactically effective amount,” or“therapeutically effective amount” refers to an amount of an agent orcomposition that provides a beneficial effect or favorable result to asubject, or alternatively, an amount of an agent or composition thatexhibits the desired in vivo or in vitro activity. “Effective amount,”“prophylactically effective amount,” or “therapeutically effectiveamount” refers to an amount of an agent or composition that provides thedesired biological, therapeutic, and/or prophylactic result. That resultcan be reduction, amelioration, palliation, lessening, delaying,prevention, and/or alleviation of one or more of the signs, symptoms, orcauses of a disease, disorder or condition in a patient/subject, or anyother desired alteration of a biological system. With regards to cellproliferation disorders, a favorable result includes reducing impacts orseverity of symptoms associated with a disease or disorder and/orincreasing life expectancy compared to that in the absence of treatment.An effective amount can be administered in one or more administrations.The relationship between the dose levels in animals and humans (based onmilligrams per square meter of body surface area) is described, forexample, in Freireich et al., (1966) Cancer Chemother Rep 50: 219.

For any composition, an effective amount can be first estimated eitherin accordance with cell culture assays or using animal models, typicallymice, rats, guinea pigs, rabbits, dogs or pigs. An animal model may beused to determine an appropriate concentration range and route ofadministration. Such information can then be used to determineappropriate doses and routes of administration for humans. Whencalculating a human equivalent dose, it is recommended to use aconversion table given in the Guidance for Industry and the Reviewersdocument (2002, US Food and Drug Administration, Rockville, Md., USA).An effective daily dose is generally 0.01 mg/kg patient weight to 2000mg/kg patient weight of an active agent, preferably 0.05 mg/kg patientweight to 500 mg/kg patient weight of an active agent. An exacteffective dose will depend on the severity of the disease, patient'sgeneral state of health, age, body weight and sex, nutrition, time andfrequency of administration, combination(s) of medicines, responsesensitivity and tolerance/response to administration and other factorsthat will be taken into account by a person skilled in the art whendetermining the dosage and route of administration for a particularpatient based on his/her knowledge of the art. Such dose may bedetermined by conducting routine experiments and at the physician'sdiscretion. Effective doses will also vary depending on the possibilityof their combined use with other therapeutic procedures, such as the useof other agents.

As used herein, a “patient” and a “subject” are interchangeable termsand may refer to a human patient/subject, a dog, a cat, a non-humanprimate, etc.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Melanoma

Melanoma is a malignant tumor that develops from melanocytes(pigment-containing cells that produce melanin), which are predominantlylocated in the basal layer of the skin's epidermis and the middle layerof the eye (Hurst E A et al., Archives of Dermatology Research, 2003,139: 1067-1073). This type of pathology accounts for 10 percent of allmalignant skin lesions. Its annual incidence rate is 5%. Starting fromthe 1940s, the incidence of melanoma has doubled every year. Melanoma isthe sixth most common cancer among men and the seventh most commoncancer in women. The average incidence rates for skin melanoma vary from3-5 cases per 100,000 people per year in the Mediterranean countries to12-20 cases per 100 thousand people per year in the Nordic countries andcontinue to grow. The death rate is 2-3 cases per 100 thousand peopleevery year with slight changes depending on the geographical location,and has remained relatively stable over the past decade. An increasedexposure to ultraviolet radiation of a genetically predisposedpopulation, at least in part, results in a steady increase in melanomaincidence over the past decades (Oncology Clinical Practice Guidelinesof the European Society for Medical Oncology (ESMO), 2010, p. 294-300).Malignant melanoma is responsible for 60-80% of deaths from skin cancersand its five-year survival rate is 14%. In the United States, 2% of thepopulation was diagnosed with this type of skin cancer, which is a causeof approximately 10,000 deaths every year. At the same time melanoma isa tumor with an extremely high potential of systemic metastases.

Primary melanoma is resistant to chemotherapy and radiation therapy. Themain method of treatment of patients with primary melanoma is surgicalremoval of the tumor and some normal tissue around it. The amount oftissue removed with surgical excision depends on the tumor thickness(Breslow thickness) and the depth of the tumor invasion. However,surgical treatment of melanoma has a number of significant disadvantagesassociated with prolonged wound healing and occurrence of implantationmetastases within 2 years of the surgery. This method of treatment isused only at early stages of the disease (stage 1-2). At the advancedstages, radiation therapy, chemotherapy, and immunotherapy areadditionally used.

Antitumor activity is exhibited by various compounds, such as alkylatingcompounds, antimetabolites, antibiotics, substances of plant or animalorigin, hormonal preparations, and enzymes, which differ in theirmechanism of action and chemical structure. However, thesechemotherapeutic drugs produce severe adverse effects on normal tissuesor organs and thus limiting their usefulness.

Possibilities for treating melanoma are limited. Recently, a number ofworks have demonstrated that an isotopic composition of tissues andorgans may serve as a diagnostic marker. In particular, a study of Cuand Zn isotope ratios in blood showed promising correlations betweenisotopic values and age, gender and pathologies. For example,determination of copper isotope ratio in blood serum is a new approachfor the diagnosis and prognosis of the development of liver cirrhosis(M. Costas-Rodriguez et al., Isotopic analysis of Cu in blood serum bymulti-collector ICP-mass spectrometry: a new approach for the diagnosisand prognosis of liver cirrhosis, Metallomics 2015, 7: 491-498). Zincisotopic composition in breast tissue can help in the diagnosis ofbreast cancer (F. Lamer et al., Zinc isotopic compositions of breastcancer tissue, Metallomics 2015, 7: 112-117). Further, certain patentsand patent applications discuss the use of isotopically enrichedcompositions for therapeutic use. See, e.g., U.S. Pat. Nos. 9,861,659;10,183,041, and 10,226,484.

WO2007/140280 suggests using an anti-cancer composition for topicaladministration comprising a cesium ion source and/or a rubidium ionsource as pharmaceutically acceptable salts to be used for melanomatreatment. The feasibility of using this therapy is based on an approachthat involves changing the acidic pH of cancer cells to slightlyalkaline, whereby the survival of cancer cells is compromised, and theformation of acidic and toxic materials, usually formed in cancer cells,is neutralized and eliminated (Sartori H E. Nutrients and cancer: anintroduction to cesium therapy, Pharmacol. Biochem. Behav. 1984; 21,Suppl. 1: 7-10). Thus mass spectrographic and isotope studies have shownthat potassium, rubidium and cesium are most effectively absorbed bycancer cells. Glucose can still enter the cell but oxygen cannot;therefore, the cell becomes anaerobic. In the absence of oxygen,fermentation of glucose to lactic acid occurs, and the pH of the cell isreduced to 7 and finally to 6.5. Cesium, rubidium and potassium, whichcreate high pH values, are able to enter the cell in such state andincrease its pH value. In this setting, cesium and rubidium ions canchange ionic physiology of the cancer cell, including inhibition of thetransmembrane movement of potassium. It is assumed that cesium andrubidium efficiently control potassium and bound hydrogen ion (H⁺)fluxes that affect all acid-dependent cancers and provide site affinityfor selective increase in the pH of the tumor microenvironment. In theauthor's opinion, this provides selective tumor modulation, but theapplication materials do not contain any information that would confirmthe effectiveness of using this composition to treat cancer. Inaddition, the composition described in this application is for topicaladministration, which cannot provide high efficacy in treating apatient. Thus the use of the described approach, although targeted oncancer cells, still is not able to provide effective eradication oftumor cells, but rather can only be used as a food supplement inaddition to other methods of treatment.

Methods

In one aspect, a method is provided for preventing the development ofmelanoma. The method effectively suppresses the development of amalignant tumor without surgical intervention and damage to surroundingnormal tissues, and additionally exhibits a high antimetastatic effect.The use of the claimed method makes it possible to achieve effectiveinhibition of the development of melanoma without producing adverseeffects on the body, as is characteristic of chemotherapeutic drugs. Themethod comprises administering to a subject in need thereof a lightisotope of zinc in the form of aspartic acid salt. A pharmaceuticalcomposition is provided to be used in the disclosed method forpreventing the development of melanoma, which comprises ⁶⁴Zn_(e)(Asp)₂in a therapeutically effective amount. The disclosed method has highefficacy in inhibiting the growth of tumor cells, along with low toxiceffect.

A method of treating melanoma or preventing the development of melanomais disclosed. The method comprises intratumoral and/or intravenousadministration of a composition that contains ⁶⁴Zn_(e)(Asp)₂ in atherapeutically effective amount. The administration of the compositionmay be single or multiple. In certain embodiments, the treatment regimencomprises 5 to 10 injections of ⁶⁴Zn_(e)(Asp)₂. In certain embodiments,the aspartate present in ⁶⁴Zn_(e)(Asp)₂ is at least 90% theL-enantiomer, at least 95%, at least 98%, or all L-isomer. In someembodiments, it may be the D-enantiomer and in other embodiments it maybe a mixture of the two enantiomers.

A method is provided of treating melanoma or preventing the developmentof melanoma comprising the administration of a composition comprising atherapeutically effective amount of ⁶⁴Zn_(e)(Asp)₂ to a patient in needthereof. In some embodiments, the composition is an aqueous solution. Insome embodiments, the composition is administered intratumorally orintravenously. In some embodiments, the ⁶⁴Zn_(e)(Asp)₂ comprises 2molecules of aspartic acid. In some embodiments, from 0.2 μg/kg patientweight/day to 2000 mg/kg patient weight/day of ⁶⁴Zn_(e)(Asp)₂ isadministered to the patient. In some embodiments, from 0.01 mg/kg/day to5 mg/kg/day of ⁶⁴Zn_(e)(Asp)₂ is administered to the patient. In someembodiments, wherein from 0.1 mg/kg/day to 1 mg/kg/day of ⁶⁴Zn_(e)(Asp)₂is administered to the patient. In some embodiments, the composition isadministered once a day. In other embodiments, the composition isadministered more than once a day. In some embodiments, the compositionfurther comprises deuterium-depleted water as a solvent. In someembodiments, the method of treating/preventing melanoma is a method thatprevents, delays, or ameliorates melanoma metastasis.

Enantiomeric purity of the aspartate in ⁶⁴Zn_(e)(Asp)₂ may be determinedby methods known in the art, such as, for example, chiralchromatography.

The presence of zinc in the ⁶⁴Zn_(e)(Asp)₂ compound or otherzinc-containing compounds may be confirmed by methods known in the art,such as, for example, atomic emission spectroscopy with an inductivelycoupled plasma. Prior to subjecting the sample to atomic emissionspectroscopy with an inductively coupled plasma, the sample may betreated with a mixture of mineral acids in a Teflon autoclave under theaction of microwave radiation.

Elemental impurities or impurities of sulfate ion in the ⁶⁴Zn_(e)(Asp)₂compound or other zinc-containing compounds may be determined by methodsknown in the art, such as, for example, atomic emission spectroscopywith an inductively coupled plasma. Prior to subjecting the sample toatomic emission spectroscopy with an inductively coupled plasma, thesample may be treated with a mixture of mineral acids in a Teflonautoclave under the action of microwave radiation.

Light isotopes may be purchased. Zn-64 oxide with the necessary degreeof enrichment may be purchased from, for example, Oak Ridge Nationallaboratory, Oak Ridge, Tenn., USA.

In certain embodiments, an effective amount of ⁶⁴Zn_(e) administered toa subject in need thereof may be from 0.2 μg/kg patient weight/day to2000 mg/kg patient weight/day. This range of from 0.2 μg/kg/day to 2000mg/kg/day corresponds to the amount of zinc present in the compositionas part of aspartate. In further embodiments, the range of ⁶⁴Zn_(e)administered is from 0.01 mg/kg/day to 5 mg/kg/day, more preferably 0.1mg/kg/day to 1 mg/kg/day. Compositions for use in the disclosed methodscontain corresponding amounts. For example, a composition for use of adisclosed method contains an amount of ⁶⁴Zn_(e) such that a single doseof the composition contains from 1 to 100 mg ⁶⁴Zn_(e) in the form of⁶⁴Zn_(e)(Asp)₂, such as 1, 5, 10, 20, 30, 40, 50, or 100 mg ⁶⁴Zn_(e). Anexemplary composition is a solution of ⁶⁴Zn_(e)(Asp)₂ that contains 1 mg⁶⁴Zn_(e) per ml of solution. In some embodiments, the solvent isdeuterium-depleted water. The solution is formulated for oral orparenteral administration, such as administration by injection, such asby intratumoral or intravenous administration. Compositions forinjection may be aqueous solutions, such as solutions with a salinityand pH optimized for the route of injection. The solution may containexcipients such as DMSO. The DMSO may be present at a concentration of1%. Another exemplary composition for used in the disclosed method is atablet or other solid composition for oral administration that contains30 mg ⁶⁴Zn_(e). The composition may also be a liquid for oraladministration, such as an aqueous composition, for example, a syrup.

A treatment regimen of the disclosed method can include eitherintratumoral administration or intravenous administration of acomposition for use in a disclosed method, or both routes ofadministration. When both routes are used in a patient, the samecomposition may be administered via both routes, or differentcompositions may be administered. In some embodiments, combinedintratumoral and intravenous administration routes are used.

In the Examples, melanoma model systems are used to study the efficacyof a composition comprising ⁶⁴Zn_(e)(Asp)₂ to simulate in vivo a numberof processes of tumor dissemination in warm-blooded animals, includingoperative/surgical intervention in case of a possible spread of tumorcells to remote sites and/or local niches. Postoperative administrationof the composition and/or its administration to prevent a possiblemelanoma metastatic process (and hence tumor progression) providessuppression of the metastatic process and has significant advantages.

Without having an intention to be associated with any scientific theory,the following explanation of the efficacy of ⁶⁴Zn_(e)(Asp)₂ is offered.Cancer tissues are largely enriched with heavy isotopes (such as Zn⁷⁰)and depleted of light isotopes (such as ⁶⁴Zn_(e)) of the basic elements.Substitution of ⁶⁴Zn_(e) by ⁷⁰Zn_(e) (⁷⁰Zn-enriched zinc) may result inan isotope-induced change in the chirality of one or more amino acids inthe protein structure and, as a result, may affect the conformation ofreceptors, ligands, and signal molecules. Loss of the structurecorrectness, depletion and degradation of proteins may lead todisruption in intra- and intercellular homeostasis, and, as aconsequence, to various pathologies. The rate of development of thedisease and symptom expression may be slow or rapid, depending on thecharacteristics of pathological chiral amplification of amino acids dueto autocatalytic reactions in living cells. Generally, the reactions ofasymmetric autocatalysis have a nonlinear character of the relationshipbetween yield and time. The most dramatic consequences occur when theabove changes take place in the p53 protein, known as the “protector ofthe genome”, two-thirds of which consist of zinc fingers. There are fivestable isotopes of zinc. In fact, “wrong” conformation of p53 causesfailures in stopping the cell cycle, malfunctions of differentiation,apoptosis, metabolism, genomic stability, angiogenesis, DNA repair,aging, and other processes. What is not known is that the very onset ofpathological changes is due to chirality induced by isotopesubstitution, leading to changes in the conformation of proteins. Thesechanges are apparently reversible, and therefore a return to the normalstate can be achieved by using light isotopes. With regard to p53, thismay be the ⁶⁴Zn isotope.

The disclosed method results in isotopic selective protein modulation,which makes it possible not only to restore damaged negative feedback inthe cell communication system by restoring transmitting and receivingreceptors and signal molecules but, more importantly, opens apossibility for eliminating mutations in biomolecules by reactivatingthe normal functions of p53 protein and associated pathways. The massspectrometric study shown in the Examples provides confirmation.

The present invention is described more fully hereinafter by referenceto the following examples, which are presented by way of illustrationonly and should not to be construed to in any way limit the scope of thepresent invention.

EXAMPLES Example 1. In Vivo Study Supporting the Efficacy of the ClaimedMethod Carried Out in Mouse Models (Intratumoral Administration of⁶⁴Zn_(e)(Asp)₂)

B16 melanoma cells having the following characteristic were used in theexperiment:

Origin: Mus musculus skin (C57BL/6 mouse)

Characteristics of tumor growth: short incubation period, rapid growth,absence of spontaneous metastases.

Tumor inoculability is 100%. The minimum dose of cells that causes tumorgrowth for this melanoma is only 100 to 1000 per subcutaneous injectionin the mouse.

An average life expectancy of animals is 21-31 days.

The tumor cell population is heterogeneous and includes both highlypigmented regions and fragments with a low content of melanin.

Karyotype: The number of chromosomes varies from 30 to 80, 2n=40, themodal number is 72 chromosomes (14%), polyploidy is 3%. All cellscontain 2 to 8 interchromosomal associations according to Robertsoniantranslocations.

A characteristic feature of B16 melanoma cells is a very low mRNAexpression level of c-myc, c-jun, and c-fos oncogenes and the absence ofc-ras, c-abl, c-erb-B2, B-lym, c-sis and c-myb oncogene expression.Another feature inherent in the cells of this melanoma is that theyproduce in vitro a large amount of the factor exhibiting procoagulantactivity.

A study of the phenotypic characteristics of melanoma cells usingimmunocytochemical analysis has shown that the cells expressVE-cadherin, N- and E-cadherins, Twist and Slug transcription factors,P-glycoprotein, ERCC, DAB2, TAP1 and weakly express CD44.

All studies that involved using mice were conducted in compliance withthe rules of the European Convention for the Protection of VertebrateAnimals used for Experimental and Other Scientific Purposes [Commissionof the European Communities: Council Directive of 18 Dec. 1986 on theLows, regulating the Application of Principles of Good LaboratoryPractice and the Verification of Their Applications for Tests onChemical Substances (87/18/EEC). The Rules Governing Medicinal Productsin the European Community.—1991.—V. 1.—P. 145-146].

The animals were selected according to the objectives of the experimentand in conformity with generally accepted requirements for preclinicalstudies of test products, including biologic drugs showing antitumoractivity.

The animals were maintained in accordance with the standards set forthin The Guide for Care and Use of Laboratory Animals (ILAR publication,1996, National Academy Press, 1996). During the experiment, the animalswere kept in plastic cages, had a natural cycle of daytime andnighttime, were given standard diet and had free access to food andwater.

Clinical Observations

All the animals in cages were examined daily in order to determinemortality or any signs of deviations in their health status. A thoroughexamination was performed each time any abnormalities were detected. Alldeviations were recorded.

Statistical processing of the results was carried out using STATISTISA6.0 software package designed for analysis in medical and biologicalstatistics using the Student's t-test; differences with a probability ofnot less than 95% were considered significant (p<0.05).

15 female C57Bl/J6 mice (5 mice per group) at the age of 10-12 weeks,weighing 18-22 g were used in the experiment.

B16 melanoma cells were cultured in vitro under standard conditions. Fortransplantation, tumor cells in the exponential phase were removed fromthe substrate with 0.02% Versene solution and the suspension cellularityand viability was evaluated in the presence of trypan blue in ahemocytometer and the suspension was adjusted to a concentration of 10⁷cells/ml by dilution with saline solution. Melanoma cells were injectedintracutaneously (i.c.) in 0.05 ml of the suspension (0.5×10⁶cells/mouse) into the animal's back region. 24 hours before theinjection of tumor cells, the wool cover on the back of each mouse wasremoved with depilation cream.

Grouping. The animals were divided into groups as follows:

Group No. 1: control group, mice injected i.c. with B16 melanoma cells;

Group No. 2: mice injected i.c. with B16 melanoma cells+intratumoralinjections of ⁶⁴Zn_(e)(Asp)₂ at day 5 after the tumor cells wereinoculated.

Group No. 3: mice injected i.c. with B16 melanoma cells+intratumoralinjections of ⁶⁴Zn_(e)(Asp)₂ at day 11 after the tumor cells wereinoculated

The composition to be administered was prepared immediately prior to itsadministration. ⁶⁴Zn_(e)(Asp)₂ was dissolved in deuterium-depleted waterwith addition of 1% DMSO. Animals in group No. 2 were injected with thecomposition comprising ⁶⁴Zn_(e)(Asp)₂ intratumorally and around the areaof tumor growth at a dose of 200 μg/mouse in a volume of 20 μl/mouseafter the tumor reached 0.5 cm in diameter (at day 5 after i.c.administration of the tumor cells). The following injection of⁶⁴Zn_(e)(Asp)₂ was given according to the above scheme in the event anexperimental animal had a new tumor growth.

Animals in group No. 3 were injected with the composition comprising⁶⁴Zn_(e)(Asp)₂ intratumorally and around the area of tumor growth at adose of 300 μg/mouse in a volume of 30 μl/mouse at day 11 after i.c.administration of the tumor cells. The follow-up injection of⁶⁴Zn_(e)(Asp)₂ was given in the event an experimental animal had a newtumor growth but at a dose 200 μg/mouse in a volume of 20 μl/mouse. 24hours after the first injection and then a series of injections weregiven every other day. ⁶⁴Zn_(e)(Asp)₂ was administered intratumorallyevery other day for 10 days, 5 times in total.

Regarding the 3rd group: group 3 mice received 300 mcg/mouse of ⁶⁴Zn_(e)in the form of ⁴Zn_(e) aspartate on the 11th day and 200 mcg/mouse of⁴Zn_(e) aspartate on the 13th, 15th, 17th and 19th days after tumor cellinjection.

Regarding the 2nd group: group 2 mice received 200 mcg/mouse of ⁴Zn_(e)aspartate on the 5th day after tumor cell injection and then ⁴Zn_(e)aspartate was given in the event an experimental animal had a new tumorgrowth at a dose 200 mcg/ml.

The anti-tumor efficacy of ⁶⁴Zn_(e)(Asp)₂ was evaluated on the basis ofthe growth dynamics of melanoma tumors estimated based on the changes intumor volume according to generally accepted rules.

From the time the experimental tumors reached 0.3-0.5 cm in diameter andevery 2-3 days thereafter, the size of each tumor node in experimentalanimals was measured in three orthogonal planes (width×length×height(W×L×H)) and the tumor volume was calculated by use of the followingformula for the volume of ellipsoid: V=4/3×πabc; where V is the tumorvolume (mm³); a, b, c is the rumor radius (mm): a is the radius alongthe x axis, b is the radius along the y axis, c is the radius along thez axis.

Statistical data processing: The Student's t-test was used to seewhether there is a statistically significant difference between themeans of the groups. Calculations were made using STATISTICA 6.0software package.

During the study of antitumor activity of the method comprisingadministration of ⁶⁴Zn_(e)(Asp)₂, results that indicate thatintratumoral administration of the composition comprising ⁶⁴Zn_(e)(Asp)₂suppresses the development of melanoma in mice (see Table 1) wereobtained. FIGS. 1-4 show the results of the efficacy of the claimedmethod in mice. The survival rates of the experimental animals thatreceived intratumoral injections of the composition comprising⁶⁴Zn_(e)(Asp)₂ were also determined during the experiment (see Table 2).

TABLE 1 Kinetics of B-16 melanoma tumor growth under the effect of⁶⁴Zn_(e)(Asp)₂ Day after inoculation of tumor cells Experimental 4 6 912 14 17 21 24 27 group Tumor volume, mm³ (M ± m) B16 melanoma 27.8 ±69.2 ± 125.4 ± 231 ± 393.3 ± 923 ± 2504 ± 3610 ± 3793.5 ± Control 3.98.6 24.3 36.8 75.5 324 758 731 495 B16 melanoma + 24.3 ± 28.2 ± 25.6 ±36.6 ± 16 ± 18.4 ± 42.6 ± 68.3 ± 155.4 ± ⁶⁴Zn_(e)(Asp)₂ 3 10^(a) 6.8^(b)20^(c) 6^(d) 12^(a) 35^(a) 54^(d) 123^(e) at day 5 after inoculation oftumor cells B16 melanoma + 27 ± 63 ± 119 ± 260 ± 184 ± 250 ± 375.4 ± 881± 2313.5 ± ⁶⁴Zn_(e)(Asp)₂ 4.2 10 26 20 51 104 135^(a) 263.6^(a) 748.5 atday 11 after inoculation of tumor cells ^(a)p < 0.05, ^(b)p < 0.02,^(c)p < 0.01, ^(d)p < 0.005, ^(e)p < 0.002 vs. control

It should also be noted that in group No. 2 (administration of thetherapeutic composition at day 5 after inoculation of tumor cells), thegrowth of melanoma was suppressed by 100% in 2 out of 5 experimentalanimals after a single intratumoral injection of ⁶⁴Zn_(e)(Asp)₂.

TABLE 2 Survival of laboratory animals (5 animals per group) under theeffect of ⁶⁴Zn_(e)(Asp)₂ Day after inoculation of tumor cellsExperimental 4 6 9 12 14 17 21 24 27 28 31 group Number of alive mice, %B16 melanoma 100 100 100 100 100 80 80 80 60 40 20 Control B16melanoma + 100 100 100 100 100 100 100 100  100  80* 80 ⁶⁴Zn_(e)(Asp)₂at day 5 after inoculation of tumor cells B16 melanoma + 100 100 100 80⁺ 80 80 80  60* 60 40 40 ⁶⁴Zn_(e)(Asp)₂ at day 11 after inoculationof tumor cells *necrosis, ulcer; ⁺the animal died immediately after theinjection because of too high dose of the composition.

TABLE 3 Growth kinetics of B16 melanoma during ⁶⁴Zn_(e)(Asp)₂ therapy ingroup 2 and individual regimens of drug administration Group and Dayafter tumor cell inoculation tumor volume 4 day 5 day 6 day 10 day 12day 13 day 14 day 17 day 18 day 21 day Melanoma 33.5 62 94.2 199.4 342 —— B16 24.4 102 211 377 692.7 1854.6 4456.5 Control 14.1 65.4 121 200.6282.7 864 1466 33.5 65.4 134 201.6 322.5 550 2937.4 33.5 51 67 176 326.7424 1157 Melanoma 14.1 Tumor+/ 14.1 24.4 24.4 — 14 4.2 — 0 B16 + 1st⁶⁴Zn_(e)(Asp)₂ injection IC on day 5 33.5 Tumor+/ 33.5 24.4 14 — 14 4.2— 0 1st injection 24.4 Tumor +/ 14.1 14 14 — 4.2 4.2 — 0 1st injection25 Tumor+/ 65.4 51 116.4 Tumor+/ 38.4 65.4 — 179.6 1st 2nd injectioninfection 24.4 Tumor+/ 14.1 14 14 Eschar 9.2 14 Tumor+/ 33.5 1st 2ndinjection injection Group and Day after tumor cell inoculation tumorvolume 22 day 24 day 25 day 28 day 36 day 39 day 43 day 55 day 58 day 61day Melanoma — — B16 5026.5 — Control 2246.2 3298.7 4712.4 — 2456.74288.3 Melanoma — 25.4 Tumor+/ 33.2 Tumor+/ Tumor+ Tumor+ 4th Tumor+/Died* B16 + 2nd 3rd injection Tumor ⁶⁴Zn_(e)(Asp)₂ injection injectionnecrosis IC on day 5 — 0 — 0 Tumor− Tumor− Tumor− Tumor− Tumor− Tumor−Tumor− — 0 — 0 Tumor− Tumor− Tumor− Tumor− Tumor− Tumor− Tumor− Tumor+/282.7 Tumor+/ 641 — — — — — 3rd Tumor Died* injection necrosis — 33.5 —102.6 Tumor+/ Tumor+/ Died* — — Tumor+/ 4th Tumor 3rd injection necrosisinjection Tumor− absence of tumor growth Tumor+ presence of tumor

As can be seen from the presented data, intratumoral administration ofZn_(e)(Asp)₂ on the 5th day after inoculation of tumor cells resulted ina significant reduction in tumor volume (the tumor volume inexperimental animals treated with ⁶⁴Zn_(e)(Asp)₂ was 4% of thecorresponding figure in the control group). The antitumor effect of⁶⁴Zn_(e)(Asp)₂ in the group where drug administered on the 11th dayafter inoculation of tumor cells first was less pronounced.Statistically significant suppression of tumor growth was observed onlyon the 21st and 24th days of the experiment. The size of melanoma wasreduced by 85% (21st day) and by 75.6% (24th day) compared to control.Antitumor activity against melanoma also manifested itself in thepercentage of alive animals on the 31st day. In particular, the survivalrate for mice injected with ⁶⁴Zn_(e)(Asp)₂ at day 5 after inoculation oftumor cells was 80%, while only 20% of animals survived in the controlgroup.

Table 3 shows growth kinetics of B16 melanoma during ⁶⁴Zn_(e)(Asp)₂therapy in group 2 and individual regimens of drug administration

Example 2. In Vivo Study Supporting the Efficacy of the Claimed MethodCarried Out in Mouse Models (Intravenous/Intravenous+IntratumoralAdministration of ⁶⁴Zn_(e)(Asp)₂)

An experimental model of hematogenous metastasis was used in theexperiment which allowed for the inoculation of B16 melanoma cells. Thecharacteristic of these cells is given in Example 1. The cells werecultured in vitro under standard conditions. For transplantation, tumorcells were removed from the substrate with 0.02% Versene solution andthe suspension cellularity and viability were evaluated in the presenceof trypan blue in a hemocytometer and the suspension was adjusted to aconcentration of 1×10⁶ cells/ml. Melanoma cells were injectedintracutaneously (IC) in 0.05 ml of suspension (0.5×10⁶ cells/mouse)into the back area of the animal. Part of the coat on the back of micewas removed with a depilatory cream at 24 hours before tumor cellinoculation. Cells isolated from B16 mouse melanoma were used in theexperiment. Cells were cultured in vitro under standard conditions. Forinoculation, tumor cells were removed from the substrate with 0.02%Versene solution. The cellularity and viability of the obtained cellsuspension were evaluated with a hemocytometer in presence of a trypanblue. Cell concentration was adjusted with saline to 1×106 cells/ml.Melanoma cells were injected IV in 0.2 ml of suspension (0.2×10⁶cells/mouse) into the lateral tail vein.

⁶⁴Zn_(e)(Asp)₂ was dissolved in deuterium-depleted water. Thecomposition was injected intravenously using a microinjection syringe ata dose of 60 μg of ⁶⁴Zn_(e)/mouse: two injections at a dose of 30μg/mouse, each in a volume of 0.3 ml (0.6 ml in total), for 4 hours. Theinjections were given every other day for 10 days (5 injections intotal). The first injection of the inventive composition was given 45minutes or 24 hours after inoculation of tumor cells. At day 26 after IVinjection of tumor cells the lungs were excised from all animals in eachgroup and the number and volume of metastases were then determined.C57Bl mice (8 mice per group) at the age of 12-14 weeks, weighing 25-27g were used in the experiment. The conditions in which animals weremaintained are described in Example 1 above.

Before the experiment, all the animals were healthy, with normalbehavioral performance. During the experiment, the animals weremaintained in plastic cages under natural light illumination on astandard diet with free access to food and water.

The ⁶⁴Zn_(e)(Asp)₂ composition was injected intravenously into theanimal's lateral tail vein using a Micro-Fine Plus microinjectionsyringe (Becton Dickinson). The injection site was cleaned with 96%ethanol.

All the animals in cages were examined daily in order to determinemortality or any signs of deviations in their health status. A thoroughexamination was performed each time any abnormalities were detected. Alldeviations were recorded.

Statistical processing of the results was carried out using STATISTICA6.0 software package designed for analysis in medical and biologicalstatistics using the Student's t-test.

The animals were divided into experimental groups as follows:

Group No. 1: control group, mice injected IV with B16 melanoma cells+IVinjection of the solvent (deuterium-depleted water);

Group No. 2: mice injected IV with B16 melanoma cells+IV injection of⁶⁴Zn_(e)(Asp)₂ 24 hours after the tumor cells were inoculated;

Group No. 3: mice injected IV with B16 melanoma cells+IV injection of⁶⁴Zn_(e)(Asp)₂ 45 minutes after the tumor cells were inoculated

The growth of metastases was assessed as follows.

On day 26 after IV injection of tumor cells the lungs were excised fromall animals in each group and the number and volume of metastases werethen determined.

The volume of metastases was calculated using the following formula forthe volume of a sphere:

V=4/3×πr³, where V is the metastasis volume (mm³); r is the metastasisradius (mm).

To assess the in vivo antimetastatic activity of ⁶⁴Zn_(e)(Asp)₂ in theexperimental model of hematogenous B16 melanoma metastasis, data on thenumber and volume of metastases in the lungs of mice were used. Theresults of the experiment are shown in Table 4 and in FIGS. 1, 2, 3, 4A,4B, 4C, 5A and 5B.

TABLE 4 Characteristics of metastatic activity of B16 melanoma cellsunder the effect of ⁶⁴Zn_(e)(Asp)₂ Number of Number of Volume of animalsin metastases/ metastases/ the group mouse mouse, with Experimentalgroup (M ± m) mm³ (M ± m) metastases, % B16 control 18 ± 4.8 15.8 ± 5.4 100 B16 + IV injection 6.4 ± 2.5  3.3 ± 2* 75 of ⁶⁴Zn_(e)(Asp)₂ at adose of 60 μg/mouse 24 hours after the tumor cells were inoculated B16 +IV injection  4 ± 2*  2.5 ± 1.7* 75 of ⁶⁴Zn_(e)(Asp)₂ at a dose of 60μg/mouse 45 min after the tumor cells were inoculated *p < 0.05 vs.control M—the arithmetic mean, and m—standard error of the mean M (i.e.,standard error of the M value)

The above results confirm the efficacy of ⁶⁴Zn_(e)(Asp)₂ in suppressingthe development of metastatic process of melanoma. A statisticallysignificant decrease in the number and volume of metastases after theuse of selected routes of administration was demonstrated. The effectwas more pronounced, both in volume and in the number of metastaticlesions of the lung tissue, where the injection was given within ashorter time (45 min) after the tumor cells were inoculated, whichindicates good prospects and the need to shorten the time between theonset of tumor growth, the spread of malignant cells and the beginningof therapy with ⁶⁴Zn_(e)(Asp)₂.

A possibility of simultaneous intratumoral and intravenousadministration of the composition comprising ⁶⁴Zn_(e)(Asp)₂ was assessedin additional experiments (data not shown). These experiments confirmedthe efficacy of ⁶⁴Zn_(e)(Asp)₂ in suppressing the metastatic process inthe body, which displayed itself in a decrease in tumor size, anincrease in the number of surviving animals, and a decrease in thenumber of metastases.

Example 3. Study of the Distribution of Light and Heavy Isotopes ofChemical Elements in Samples of Cutaneous Melanoma

The distribution of isotopes of various chemical elements in samples ofcutaneous melanoma from experimental animals and corresponding samplestaken from healthy animals was analyzed and compared in the experiment.Glow discharge mass-spectrometry was used for the determination of traceelements. The samples for the analysis were prepared as follows.

1. A sample of prepared tissue weighing up to 1 g was submerged inliquid nitrogen. With superfast cooling at a speed of about 100° per 1sec, most of the water turns into amorphous ice, the structure of whichdiffers little from the structure of water and does not undergovolumetric expansion. Due to this fact, the structure of the tissue,after being frozen, does not change at the cellular level.

2. Amorphous ice was removed by sublimation under vacuum at a lowtemperature with automatic supply of dry nitrogen to the drying chamberto accelerate the sublimation process. The volume of gas supplied to thedrying chamber did not exceed 0.11/min. The drying time of the sampleunder these conditions was about 10 hours.

3. Secondary drying was carried out under vacuum. To this end, at astage of almost complete (up to 99%) dehydration, the sample was heatedto a temperature of 35-40° C. under vacuum and isothermally held underthese conditions for about 1 hour.

A constant mass of the sample until two identical mass values wereobtained was a criterion for the completion of the drying process.During the last 3 hours of the drying process, the biomaterial samplewas removed from the vacuum chamber every hour and weighed on ananalytical balance. The drying process was stopped as soon as twoidentical values of the mass were obtained.

After completion of the sublimation process, the dry sample was removedfrom the vacuum chamber and laid in thin layers (not more than 5 μm eachlayer) between copper grids fixed in the cage. 50 semi-circular coppergrids 50-100 microns thick were used which were tightly pressed togetherby a metal clamp holding them. The “sandwich” prepared in this way formass spectrometric analysis consisted of 50 copper grids, between whichthe test material was firmly pressed.

The analyzed area on the clamp surface was a circle 10 mm in diameter,in the center of which there were copper grids with the sample pressedin between them.

Finnigan ELEMENT GD glow discharge mass-spectrometer with the followingspecifications was used in the experiment:

-   -   Dynamic range >10¹² linear with automatic cross calibration        (from matrix elements (100%) to ultra-traces (ppt);    -   Sensitivity (peak height, total ion current): >1×10¹⁰ cps,        1.6*10⁻⁹ A;    -   Dark Noise <0.2 cps    -   Mass resolution >10 000;    -   Mass stability 25 ppm/8 hour

Results of the study are shown in Table 5.

TABLE 5 Distribution of isotopes of chemical elements in samples ofmouse cutaneous melanoma Control Ele- Iso- Value Mean (normal ment tope1 2 3 4 5 6 value tissue) Mg Mg24 74.93 75.07 75.02 79.91 73.40 75.8075.69 78.99 Mg25 10.98 10.63 11.15 9.16 10.25 8.54 10.12 10.00 Mg2613.09 14.30 13.78 14.66 14.43 14.87 14.19 11.01 Si Si28 91.92 90.9194.12 93.48 91.15 90.32 91.98 92.23 Si29 5.56 6.06 3.53 3.62 5.51 6.455.12 4.67 Si30 2.53 3.03 2.35 2.90 3.34 3.23 2.90 3.10 Cl C135 74.6675.52 77.27 74.94 76.10 75.34 75.64 75.77 C137 25.34 24.48 22.73 25.0623.90 24.66 24.36 24.23 Ca Ca40 96.94 96.94 96.94 96.94 96.94 96.9496.94 96.94 Ca42 0.71 0.68 0.67 0.69 0.69 0.72 0.69 0.65 Ca43 0.14 0.130.14 0.13 0.13 0.13 0.13 0.14 Ca44 0.03 0.06 0.06 0.06 0.05 0.02 0.052.09 Ca46 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Ca48 2.19 2.19 2.192.19 2.19 2.19 2.19 0.19 Fe Fe54 2.84 2.27 2.95 2.31 2.60 2.74 2.62 5.80Fe56 91.86 91.61 91.47 91.52 91.33 91.81 91.60 91.72 Fe57 4.01 5.84 5.205.90 5.90 6.17 5.50 2.20 Fe58 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 ZnZn64 44.32 44.21 43.36 43.73 43.79 43.32 43.79 48.60 Zn66 26.56 27.8829.55 30.05 27.61 27.13 28.13 27.90 Zn67 6.55 6.26 7.37 7.62 7.52 7.847.20 4.10 Zn68 21.97 21.04 21.12 20.49 20.57 20.11 20.88 18.80 Zn70 0.600.60 0.60 0.60 0.60 0.60 0.60 0.60 Rb Rb85 55.00 47.22 42.42 34.48 33.3335.14 41.27 72.16 Rb87 45.00 52.78 57.58 65.52 66.67 64.86 58.73 27.84

As can be seen from the table above, the results confirm our hypothesisthat pathological changes due to chirality can be induced by isotopicsubstitutions. Thus the administration of ⁶⁴Zn_(e)(Asp)₂ according tothe present invention can prevent and restore the normal isotopedistribution necessary for the successful functioning of proteins, suchas a transcription factor or a protein that controls the cell cycle, andthereby suppress formation of malignant tumors.

Example 4

For the experiment, ⁶⁴Zn_(e)(Asp)₂ was synthesized from ⁶⁴Zn_(e) oxideand was in a powdered form after the synthesis. A solution of theconcentration required for the experiment was prepared immediately priorto its administration to the animals by dissolving the required amountof obtained ⁶⁴Zn_(e)(Asp)₂ powder in physiological saline or indeuterium-depleted water.

The method of atomic emission spectroscopy with an inductively coupledplasma was endured to confirm the presence of Zinc cation in the sample.Prior to determination the sample was treated with a mixture of mineralacids in a Teflon autoclave under the action of microwave radiation. Thepresence of Zinc in the sample was confirmed as the main cation: itsapproximate content was 17.6%, The impurities of other element s wasalso revealed.

Determination of Zn content also was made with complexometric titrationmethod with eriochrome black T as an end-point indicator. It was foundthat content of zinc in the sample to be 17.98±0.17%.

The confirmation the presence of aspartate ion and determination of itsoptical isomer form was carried out by liquid chromatography withdetection at 254 nm wavelength using chiral chromatography columnChiralcel OD-R. Previously, derivatization was carried out usingisophenyl cyanate. Also the chromatograms of derivatized standards ofthe racemic mixture D, L-aspartic acid. and pure L-aspartic acid wereobtained. It was shown that sample contains L-isomer only.

A zinc content was found to be 17.98±0.17% corresponding to 90.8±0.9% ofZinc L-aspartate.

Determination of the relative content of ⁶⁴Zn in the sample wasperformed after dissolution of sample pail in deionized water anddilution to a concentration of 1.33 ppm based on zinc. Ratio of signalintensities of ⁶⁴Zn, ⁶⁶Zn, ⁶⁷Zn, ⁶⁸Zn, and ⁷⁰Zn isotopes were measuredfor sample solution with Agilent 7500ce inductively coupled plasmamass-spectrometer. The results are shown in Table 6. The sample wasenriched for ⁶⁴Zn, measured at 99.39%.

TABLE 6 Isotope distribution Signal Rational in nature, rational Isotopeintensity, cps content % content % * ⁶⁴Zn 17 811 884.90    99.39 49.17⁶⁶Zn 70 776.20 0.3949 27.73 ⁶⁷Zn  8 779.26 0.0490 4.04 ⁶⁸Zn 27 976.100.1561 18.45 ⁷⁰Zn  1 184.63 0.0066 0.61

Sulfate ion content in the sample solution was determined byturbidimetry method using Barium chloride. The content of sulfates inthe sample was found to be 0.66±0.032%.

Elemental impurities and impurities of sulfate ion in the sample weredetermined. Elemental impurities were recovered by atomic emissionspectroscopy with an inductively coupled plasma. Prior to determination,the sample was treated with a mixture of mineral acids in a Teflonautoclave under the action of microwave radiation. Impurities ofPhosphorus, Calcium, Sodium, Silver, Aluminum, Bismuth, Copper, Ferum,Potassium, Magnesium and Plumbum were detected, as shown in Table 7.

TABLE 7 Impurity Content, % P 0.1 Ca 0.08 Na 0.05 Bi 0.01 Cu 0.01 Fe0.01 Ag 0.01 Al 0.01 K 0.01 Mg 0.01 Pb 0.0054

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of theappended claims. Thus, while only certain features of the invention havebeen illustrated and described, many modifications and changes willoccur to those skilled in the art. It is therefore to be understood thatthe appended claims are intended to cover all such modifications andchanges as fall within the true spirit of the invention.

1. A method of treating melanoma in a patient in need thereof,comprising administering a composition comprising a therapeuticallyeffective amount of ⁶⁴Zn_(e)(Asp)₂ to said patient; wherein thecomposition is administered intratumorally or intravenously, or both. 2.The method of claim 1, wherein the composition is an aqueous solution.3. (canceled)
 4. The method of claim 1, wherein ⁶⁴Zn_(e)(Asp)₂ comprises2 molecules of asp artic acid.
 5. The method of claim 1, wherein from0.2 μg/kg patient weight/day to 2000 mg/kg patient weight/day of⁶⁴Zn_(e)(Asp)₂ is administered to the patient.
 6. The method of claim 5,wherein from 0.01 mg/kg/day to 5 mg/kg/day of ⁶⁴Zn_(e)(Asp)₂ isadministered to the patient.
 7. The method of claim 5, wherein from 0.1mg/kg/day to 1 mg/kg/day of ⁶⁴Zn_(e)(Asp)₂ is administered to thepatient.
 8. The method of claim 1, wherein the composition isadministered once a day.
 8. The method of claim 1, wherein thecomposition is administered more than once a day.
 9. The method of claim1, wherein the composition further comprises deuterium-depleted water asa solvent.
 10. The method of claim 1, wherein the method of treatingmelanoma is a method that prevents, delays, or ameliorates melanomametastasis.
 11. The method of claim 1, wherein the compositionconsisting essentially of ⁶⁴Zn_(e)(Asp)₂.